JP5736230B2 - Microchip - Google Patents

Description

  The present invention includes a fluid circuit therein, and a specimen or a liquid such as a reagent existing in the fluid circuit is moved to a desired position in the fluid circuit by applying a centrifugal force, thereby testing or analyzing the specimen. The present invention relates to a microchip capable of performing the above.

  In recent years, the importance of detecting, detecting or quantifying biological substances such as DNA (Deoxyribo Nucleic Acid), enzymes, antigens, antibodies, proteins, viruses and cells, and chemical substances in fields such as medicine, health, food, and drug discovery There have been proposed various biochips and microchemical chips (hereinafter collectively referred to as microchips) that can be easily measured.

  Microchips can perform a series of analysis or experiment operations performed in the laboratory within a chip of several cm square and a thickness of about several millimeters to 1 cm. There are many advantages such as a high reaction rate, high-throughput testing, and the ability to obtain test results immediately at the site where the sample is collected. Such a microchip is suitably used for biochemical tests such as blood tests.

  A microchip is called a fluid circuit (or a microfluidic circuit), which includes a plurality of types of parts (chambers) for performing specific processing on liquids such as specimens and reagents existing in the circuit, and these parts. 2. Description of the Related Art Conventionally, a flow path network composed of fine flow paths that are appropriately connected is provided therein. In inspection or analysis of a sample using a microchip having such a fluid circuit inside, the fluid circuit is used to measure the sample introduced into the fluid circuit and the reagent mixed therewith (that is, , Movement to the weighing part which is a part for measuring), mixing of the specimen and liquid reagent (that is, movement to the mixing part which is a part for mixing them), from one part to another part Various processes such as movement are performed. Hereinafter, processing performed on various liquids (specimen, a specific component in the specimen, a liquid reagent, or a mixture of two or more thereof) performed in the microchip is also referred to as “fluid processing”. . These various fluid treatments can be performed by applying a centrifugal force in an appropriate direction to the microchip.

  In the microchip that performs fluid processing by moving the liquid in the fluid circuit to a desired position (part) in the fluid circuit using the centrifugal force as described above, when the wettability of the liquid is relatively high, There has been a problem that unintentional liquid movement occurs due to the liquid traveling along the inner wall of the fluid circuit due to the surface tension. For example, even though no centrifugal force is applied, the liquid reagent sometimes flows out from the reagent holding part, which is a part for storing the liquid reagent, along the inner wall of the flow path.

  Patent Document 1 discloses a microchip provided with a valve capable of preventing liquid discharge. However, this valve has a relatively complicated structure and has room for improvement.

JP 2007-285792 A

  An object of the present invention is to provide a microchip capable of preventing unintentional movement of liquid due to surface tension in a microchip that moves liquid existing in a fluid circuit to a desired position in the fluid circuit by applying centrifugal force. It is to provide.

  The present invention includes a fluid circuit including a space formed therein, and a microchip that moves a liquid existing in the fluid circuit to a desired position in the fluid circuit. The fluid circuit passes the liquid. A first channel that includes a first channel and a second channel through which the liquid that has passed through the first channel is passed, the first channel being an end on the second channel side Provides a microchip disposed so as to be separated from the inner wall surface of the second flow path.

  In one preferred embodiment of the present invention, the fluid circuit includes a reagent holding unit that stores a liquid reagent, and the reagent holding unit is the first end, for discharging the liquid reagent from the reagent holding unit. It has a discharge port.

  The first flow path is preferably arranged such that the first end is located in the second flow path. Moreover, it is preferable that the cross-sectional area of a 1st flow path is smaller than the cross-sectional area of a 2nd flow path.

  According to the present invention, in a microchip that moves a liquid existing in a fluid circuit to a desired position in the fluid circuit by applying a centrifugal force, it is possible to effectively prevent unintended liquid movement due to surface tension.

It is the perspective view and sectional drawing which show notionally the 1st flow path and 2nd flow path which the fluid circuit of the microchip concerning this invention has. It is sectional drawing which shows typically the mode of movement of the liquid reagent accommodated in the reagent holding | maintenance part which the microchip concerning this invention has, its vicinity, and a reagent holding part. It is sectional drawing and a perspective view which show typically the mode of the movement of the reagent holding part which the conventional microchip has, its vicinity, and the liquid reagent accommodated in the reagent holding part. It is an outline drawing which shows an example of the microchip of this invention. It is a top view which shows the 2nd board | substrate which comprises the microchip shown by FIG. It is a bottom view which shows the 2nd board | substrate which comprises the microchip shown by FIG. FIG. 5 is a top view, a cross-sectional view, and a bottom view showing a reagent holding portion and a structure in the vicinity thereof that the microchip shown in FIG. It is the top view, sectional drawing, and bottom view which show the structure of the reagent holding part which the conventional microchip has, and its vicinity. The whole blood measurement of the fluid processing using the microchip shown in FIG. 4, the liquid state on the upper surface (first substrate side surface) of the second substrate and the lower surface (third substrate side surface) in the reagent measurement step It is a figure which shows the state of a liquid. The state of the liquid on the upper surface (first substrate side surface) and the state of the lower surface (third substrate side surface) of the second substrate in the whole blood transfer step of fluid processing using the microchip shown in FIG. FIG. The liquid state on the upper surface (first substrate side surface) of the second substrate and the liquid state on the lower surface (third substrate side surface) in the blood cell separation step of fluid processing using the microchip shown in FIG. FIG. The state of the liquid on the upper surface (first substrate side surface) of the second substrate and the state of the liquid on the lower surface (third substrate side surface) in the plasma component measurement step of fluid processing using the microchip shown in FIG. FIG. The liquid state and the lower surface (third substrate side surface) of the upper surface (first substrate side surface) of the second substrate in the first step of the first mixing step of the fluid processing using the microchip shown in FIG. It is a figure which shows the state of a liquid. The liquid state and the lower surface (third substrate side surface) of the upper surface (first substrate side surface) of the second substrate in the second step of the first mixing step of the fluid processing using the microchip shown in FIG. It is a figure which shows the state of a liquid. The liquid state and the lower surface (third substrate side surface) of the upper surface (first substrate side surface) of the second substrate in the first step of the second mixing step of the fluid processing using the microchip shown in FIG. It is a figure which shows the state of a liquid. The liquid state and the lower surface (third substrate side surface) of the upper surface (first substrate side surface) of the second substrate in the second step of the second mixing step of the fluid processing using the microchip shown in FIG. It is a figure which shows the state of a liquid. The state of the liquid on the upper surface (first substrate side surface) of the second substrate and the state of the liquid on the lower surface (third substrate side surface) in the detection part introduction step of the fluid processing using the microchip shown in FIG. FIG. It is a figure which shows the result of a liquid reagent holding power test. It is a top view which shows another example of the microchip of this invention. FIG. 20 is a cross-sectional view schematically showing a reagent holding portion and a structure in the vicinity thereof which the microchip shown in FIG. 19 has. It is sectional drawing which shows typically the further another example of the microchip of this invention. It is sectional drawing which shows typically the further another example of the microchip of this invention. It is sectional drawing and perspective view which show typically another example of the microchip of this invention, and is a figure which shows the mode of the plasma component in the plasma introduction | transduction process of the fluid processing using this microchip. It is a figure which shows the mode of the plasma component in the plasma measurement process of the fluid processing using the microchip shown by FIG. It is a figure which shows the mode of the plasma component in the plasma discharge | emission process of the fluid processing using the microchip shown by FIG.

  The microchip of the present invention is a chip that can perform various chemical synthesis, inspection, analysis, and the like using a fluid circuit included therein. For example, the microchip is provided on a first substrate and the first substrate. It can be a laminated structure with a second substrate that is laminated and has grooves on the substrate surface. In this case, the fluid circuit of the microchip is an internal space formed by the groove and the first substrate surface.

  The microchip of the present invention includes a first substrate, a second substrate stacked on the first substrate, and provided with grooves on both surfaces of the substrate, and a second substrate stacked on the second substrate. 3 substrate may be included. In this case, the fluid circuit includes a first fluid circuit including a space formed by a groove provided on a first substrate side surface of the second substrate and a second substrate side surface of the first substrate, 3 has a two-layer structure of a second fluid circuit including a space formed by a groove provided on a first substrate side surface of the third substrate and a third substrate side surface of the first substrate. “Two layers” means that fluid circuits are provided at two different positions in the thickness direction of the microchip. Such a two-layer fluid circuit can be connected by a through-hole penetrating the first substrate in the thickness direction.

  The size of the microchip is not particularly limited, and can be, for example, about several cm to 10 cm in length and width and about several mm to several cm in thickness.

  The method for bonding the substrates together is not particularly limited. For example, among the substrates to be bonded, at least one of the bonded surfaces of the substrates is melted and welded (welding method), and bonded using an adhesive. And the like. Examples of the welding method include a method of welding by heating the substrate; a method of irradiating light such as a laser and welding by heat generated during light absorption (laser welding); and a method of welding using ultrasonic waves. Can do. Of these, the laser welding method is preferably used.

  The material of each substrate constituting the microchip of the present invention is not particularly limited. For example, polyethylene terephthalate (PET), polyethylene naphthalate (PEN), polybutylene terephthalate (PBT), polymethyl methacrylate (PMMA), polycarbonate (PC), polystyrene (PS), polypropylene (PP), polyethylene (PE), polyarylate resin (PAR), acrylonitrile-butadiene-styrene resin (ABS), styrene-butadiene resin (styrene-butadiene copolymer), chloride Vinyl resin (PVC), polymethylpentene resin (PMP), polybutadiene resin (PBD), biodegradable polymer (BP), cycloolefin polymer (COP), polydimethylsiloxane (PDMS), polyacetal POM), polyamide (PA) organic material (thermoplastic resin, etc.); silicon, and glass, inorganic material such as quartz. Among these, it is preferable to use a thermoplastic resin in consideration of the ease of forming a fluid circuit.

  In the case where the microchip is composed of a first substrate and a second substrate having a groove on the substrate surface, the second substrate includes a portion irradiated with detection light during optical measurement. A transparent substrate is preferable. The first substrate may be a transparent substrate or an opaque substrate. However, when laser welding is performed, it is preferable to use an opaque substrate because the light absorption rate can be increased, and the substrate is a thermoplastic resin. More preferably, a black substrate is formed by adding a black pigment such as carbon black to the resin.

  When the microchip includes a first substrate, a second substrate having grooves on both surfaces of the substrate, and a third substrate, the second substrate is made an opaque substrate from the viewpoint of the efficiency of laser welding. It is preferable to use a black substrate. On the other hand, the first and third substrates are preferably transparent substrates in order to construct the detection unit. When the first and third substrates are transparent substrates, a detection unit (optical measurement cuvette) can be formed from the through holes provided in the second substrate and the transparent first and third substrates, and the microchip. Optical measurement such as detecting the intensity (transmittance) of transmitted light by irradiating the detection unit with light from a direction substantially perpendicular to the surface can be performed.

  The method for forming grooves (pattern grooves) constituting the fluid circuit on the surface of the second substrate is not particularly limited, and examples thereof include an injection molding method using a mold having a transfer structure, an imprint method, and the like. it can. In the case of forming a substrate using an inorganic material, an etching method or the like can be used. The shape (pattern) of the groove is determined so as to obtain a desired fluid circuit structure.

  The microchip of the present invention allows a liquid (such as a specimen, a specific component in the specimen, a liquid reagent, and a mixture of two or more thereof) to be contained in the fluid circuit by applying centrifugal force. By moving to a desired position (part), an appropriate fluid treatment can be performed on the liquid. For this purpose, the fluid circuit includes various portions (chambers) arranged at appropriate positions, and these portions are appropriately connected through fine flow paths.

  The fluid circuit is a reagent holding unit for holding a liquid reagent for mixing (or reacting) with a sample to be tested or analyzed, for example, as the part (chamber); a sample introduced into the fluid circuit Separation unit for extracting a specific component from the sample; Sample measurement unit for measuring a sample (including a specific component in the sample; the same applies hereinafter); Reagent measurement unit for measuring a liquid reagent; A mixing unit for mixing; a detection unit (an optical measurement cuvette) for performing inspection or analysis (for example, detection or quantification of a specific component in the mixed solution) of the obtained mixed solution can be included. The method of inspection or analysis is not particularly limited. For example, a method for detecting the intensity (transmittance) of light that is transmitted by irradiating light to the detection unit that contains the liquid mixture, and the liquid mixture held in the detection unit An optical measurement such as a method of measuring an absorption spectrum can be given. The microchip of the present invention may have all of the above-mentioned exemplified portions, or may not have any one or more. Moreover, you may have site | parts other than these illustrated site | parts.

  Note that the “specimen” is a substance that is introduced into the fluid circuit and is a target of a test or analysis performed by the microchip, for example, whole blood. A “liquid reagent” is a reagent that processes a sample to be tested or analyzed by the microchip, or is mixed or reacted with the sample. Usually, a reagent holding part of a fluid circuit is used in advance before using the microchip. Built in.

  In a fluid circuit such as extraction of a specific component from a sample (separation of unnecessary components), measurement of a sample and / or a liquid reagent, mixing of a sample and a liquid reagent, introduction of the obtained mixture into a detection unit, etc. Various fluid treatments can be performed by sequentially applying a centrifugal force in an appropriate direction to the microchip. Application of centrifugal force to the microchip can be performed by placing the microchip on a device (centrifuge) that can apply centrifugal force. The centrifuge device can include a rotatable rotor (rotor) and a rotatable stage disposed on the rotor. A centrifugal force in an arbitrary direction can be applied to the microchip by placing the microchip on the stage and rotating the stage to arbitrarily set the angle of the microchip with respect to the rotor.

  Here, in the microchip of the present invention, as conceptually shown in FIG. 1, the fluid circuit passes the first flow path 1 through which the liquid passes and the liquid that has passed through the first flow path 1. The first channel 1 includes the second channel 2 and the first end 1a which is the end on the second channel 2 side is separated from the inner wall surface 2a of the second channel 2. (That is, the first end 1a is not in contact with the inner wall surface 2a of the second flow path 2). The first flow path 1 and the second flow path 2 may be flow paths that connect the above-described portions (chambers) that constitute the fluid circuit, or the portions (chambers) themselves or a part thereof. It may be. FIG. 1A is a perspective view conceptually showing a first flow path 1 and a second flow path 2 included in a fluid circuit of a microchip according to the present invention, and FIG. 1B is a sectional view thereof. is there.

  According to the microchip of the present invention having the above characteristics, it is possible to effectively prevent unintended liquid movement due to surface tension from the first end 1a in the first flow path 1. This advantageous effect will be described more specifically by taking as an example the case where the first end 1 is a discharge port for discharging the liquid reagent from the reagent holding unit. FIG. 2 is a cross-sectional view schematically showing the reagent holding unit and the vicinity thereof included in the microchip according to the present invention and the movement of the liquid reagent accommodated in the reagent holding unit. The microchip shown in FIG. 2 has a laminated structure of a first substrate 7, a second substrate 6 and a third substrate 5, and a groove provided on the surface of the second substrate 6 and the first substrate 7. As a result, a reagent holding part 4 for accommodating the liquid reagent X is formed [FIG. 2 (a)].

  In the microchip shown in FIG. 2, the end of the first flow path 1 extending from the reagent holding unit 4 (the first end 1a and the liquid reagent X discharge port) The liquid reagent X that has passed through the channel 1 is then separated from (not in contact with) any of the inner wall surface 2a of the second channel 2 that will pass through [FIG. 2 (a)]. Therefore, the liquid reagent X reaching the first end 1a is held without flowing out to the second flow path 2 side, and the unintended movement of the liquid reagent X to the second flow path 2 is prevented. [FIG. 2 (b)]. When the liquid reagent X is intentionally moved to the second channel 2, a centrifugal force is applied to the microchip.

  On the other hand, in the conventional microchip as shown in FIG. 3, the first end 1a of the first flow path 1 is in contact with the inner wall surface 2a of the second flow path 2, and the first Since the inner wall surface of the flow channel and the inner wall surface of the second flow channel are continuous [FIG. 3 (a)], the liquid reagent X that has reached the first end 1a is left as it is due to the surface tension. It flows out into the flow path 2 [FIG. 3 (b)]. In addition, FIG.3 (c) is a schematic perspective view of the part A shown by Fig.3 (a).

Hereinafter, the present invention will be described in more detail with reference to embodiments.
<First Embodiment>
4A and 4B are outline views showing an example of the microchip of the present invention. FIG. 4A is a top view, FIG. 4B is a side view, and FIG. 4C is a bottom view. A microchip 100 shown in FIG. 4 includes a first substrate 101 that is a transparent substrate, a second substrate 102 that is a black substrate, and a third substrate 103 that is a transparent substrate in this order [FIG. (See (b)). The vertical and horizontal lengths of these substrates are not particularly limited, but in this embodiment, the horizontal (A in FIG. 4A) is approximately 62 mm × vertical (B in FIG. 4A) is approximately 30 mm. In the present embodiment, the thicknesses of the first substrate 101, the second substrate 102, and the third substrate 103 (C, D, and E in FIG. 4B, respectively) are about 1.6 mm and about 9 mm, respectively. About 1.6 mm. However, the size of the microchip according to the present invention is not limited to the above size.

  The first substrate 101 has a reagent inlet 110 (total 11 in this embodiment) penetrating in the thickness direction and a specimen inlet 120 for introducing specimens (for example, whole blood) into the fluid circuit of the microchip. Is formed. The microchip 100 of the present embodiment is usually put into actual use after injecting a liquid reagent from the reagent inlet 110 and then sealing the reagent inlet 110 with a sealing label or the like.

  The second substrate 102 is formed with grooves formed on both sides thereof and a plurality of through holes penetrating in the thickness direction, and by bonding the first substrate 101 and the third substrate 103 thereto, A two-layer fluid circuit is formed inside the microchip. Hereinafter, a fluid circuit including the first substrate 101 and a groove provided on the surface of the second substrate 102 on the first substrate 101 side is referred to as a “first fluid circuit” and a third substrate 103. And a groove formed on the surface of the second substrate 102 on the third substrate 103 side is referred to as a “second fluid circuit”. These two fluid circuits are connected by a through hole formed in the second substrate 102 that penetrates in the thickness direction. Hereinafter, the configuration of fluid circuits (grooves) formed on both surfaces of the second substrate 102 will be described in detail.

  5 and 6 are a top view and a bottom view of the second substrate 102 included in the microchip shown in FIG. 4, respectively. FIG. 5 shows an upper fluid circuit (first fluid circuit) of the second substrate 102, and FIG. 6 shows a lower fluid circuit (second fluid circuit). In FIG. 6, the lower fluid circuit of the second substrate 102 is shown in a horizontally inverted state so that the correspondence with the upper fluid circuit shown in FIG. 5 can be clearly understood. The microchip 100 of the present embodiment is a multi-item chip that can perform six items of inspection or analysis on one specimen, and its fluid circuit has six types so that six items of inspection or analysis can be performed. It is divided into sections (sections 1 to 6 in FIG. 5) (however, these sections are connected to each other in the first component metering portion installation region (lower fluid circuit upper region)).

  In each of the above sections, one or two reagent holding units each containing a liquid reagent are provided in the first fluid circuit (upper fluid circuit) (reagent holding units 301a, 301b, and 302a in FIG. 5). , 302b, 303a, 303b, 304a, 304b, 305a, 305b and 306a in total 11). The sample introduced from the sample introduction port 120 in FIG. 4 is weighed, and after the blood cell component is separated and removed, it is distributed to each section and weighed. It is mixed with two types of liquid reagents and introduced into the detection units 311, 312, 313, 314, 315, and 316, respectively. The mixed liquid introduced into each detection unit of each section is used for optical measurement such as irradiating light to the detection unit from a direction substantially perpendicular to the microchip surface and measuring the transmittance of the transmitted light. The specific component in the mixed solution is detected. In a series of these fluid treatments, a centrifugal force in an appropriate direction is applied to the microchip, whereby a liquid reagent, a specimen, a specific component in the specimen, or a mixture of the specific component and the liquid reagent is applied to each section. It is performed by moving to each part in the provided two-layer fluid circuit in an appropriate order. The application of the centrifugal force to the microchip can be performed, for example, by placing it on the above-described centrifuge.

  Each reagent holding unit is connected to the reagent measuring unit via a flow path (through hole) that penetrates the second substrate 102. For example, the reagent holding unit 301a (see FIG. 5) and the reagent measuring unit 411a (see FIG. 6) of section 1 are connected by a flow path 21b. The same applies to other reagent holding units and reagent measuring units.

  Each section includes a component measuring unit (sample measuring units 401, 402, FIG. 6) that measures a specific component (for example, a plasma component) separated from a sample in the second fluid circuit (lower fluid circuit). 403, 404, 405, 406 in total) and a reagent metering unit for metering a liquid reagent (reagent metering units 411a, 411b, 412a, 412b, 413a, 413b, 414a, 414b, 415a, 415b and 416a in FIG. 6) 11 pieces in total) are provided. Each sample measuring section is connected in series by a flow path (see FIG. 6).

  The microchip 100 separates an unnecessary component from the sample measuring unit 500 (see FIG. 5), the flow rate limiting unit 700 (see FIG. 6), and the sample that has been introduced into the microchip. A separation unit 420 for taking out components (components to be mixed with the liquid reagent) is provided (see FIG. 6). Extraction of specific components is performed by centrifugation. The sample measuring unit 500 and the flow rate limiting unit 700 are connected by a flow path (through hole) 30.

  Further, as shown in FIG. 5, the microchip 100 includes overflow liquid storage units 330a and 330b for storing a sample or a specific component overflowing from the sample measurement unit and the component measurement unit at the time of measurement, and at the time of measurement. Are provided with overflow reagent storage portions 331a, 331b, 332a, 332b, 333a, 333b, 334a, 334b, 335a, 335b and 336a for storing the liquid reagent overflowing from the reagent metering portion. The overflow liquid storage unit 330b is connected to the component measuring unit 406 via the flow channel 16a (see FIG. 6), the flow channel (through hole) 26a penetrating in the thickness direction, and the flow channel 16b (see FIG. 5). Each overflow reagent storage unit is connected to a corresponding reagent metering unit via a flow path. For example, in section 1, the reagent measuring unit 411a for measuring the liquid reagent stored in the reagent holding unit 301a and the overflow reagent storage unit 331a (see FIG. 3) for storing the overflowing liquid reagent are They are connected via a path 11a (see FIG. 6), a flow path (through hole) 21a penetrating in the thickness direction, and a flow path 11b (see FIG. 5). The same applies to other overflow reagent storage units.

  As described above, the microchip includes the overflow liquid storage unit and the overflow reagent storage unit (hereinafter sometimes collectively referred to as the overflow storage unit), thereby detecting the presence or absence of overflow in the overflow storage unit. Thus, it is possible to easily confirm whether the specimen, the specific component, or the liquid reagent has been reliably transferred to the measuring unit by the centrifugal operation and the measuring unit is filled with the object to be measured. That is, if it is detected that the overflowing material is present in the overflow container, it is guaranteed that the sample, the specific component, or the liquid reagent is accurately measured in the measuring unit. Thereby, the reliability of inspection or analysis can be improved.

  As a method for detecting whether or not the overflow material is present in the overflow container, for example, the overflow container is irradiated with light from the first substrate 101 side which is a transparent substrate, and the reflected light A method for measuring the strength can be preferably used. The light to be used is not particularly limited, and may be monochromatic light (for example, laser light) having a wavelength of about 400 to 1000 nm, or may be mixed light such as white light. The intensity of reflected light can be measured using, for example, a commercially available reflection sensor.

  In the method of detecting the presence or absence of the overflow by measuring the reflected light intensity, basically, before the overflow is introduced into the overflow container, the first substrate is placed on the overflow container. Reflected light intensity obtained by irradiating light from the 101 side, and reflection obtained by irradiating light from the first substrate side to the overflow housing part after an object to be weighed is introduced into the weighing part. The ratio with the light intensity is obtained, and the presence or absence of overflow is detected from the intensity ratio. That is, if the ratio (reflected light intensity after introduction / reflected light intensity before introduction) is smaller than 1 (if the reflected light intensity after introduction is smaller), it is determined that there is an overflow in the overflow container. Is done. However, if the manufacturing fluctuation between microchips is small and the reflected light intensity before introducing the overflow can be regarded as almost constant between the microchips, the measurement of the reflected light intensity before introducing the overflow should be omitted. Is possible.

  Here, in the microchip 100 of the present embodiment, each reagent holding unit and the structure in the vicinity thereof have the above-described characteristics according to the present invention. The reagent holding unit 306a will be described as an example. FIG. 7 is a top view, a cross-sectional view, and a bottom view showing the structure of the reagent holding unit 306a and the vicinity thereof. FIG. 7 (a) is a top view, FIG. 7 (c) is a bottom view (shown in a horizontally reversed state with respect to FIG. 6), and FIG. 7 (b) is shown in FIGS. It is sectional drawing in the dotted line shown by c). In this cross-sectional view, the first substrate 101 and the third substrate 103 which are arranged above and below the second substrate 102 are shown together.

  As shown in FIG. 7, the reagent holding unit 306a has one end (second end) connected to the reagent holding unit 306a, and guides the liquid reagent in the reagent holding unit 306a to the reagent measuring unit 416a. The flow path (through hole) 22b is provided. This flow path 22b corresponds to the first flow path described above. Referring to FIG. 7 (b), the flow path 22b has a first end 1a (liquid reagent outlet) which is the other end of the second flow path 2 on the second fluid circuit side. It is arranged so as to be separated from any of the wall surfaces 2a (that is, the first end 1a is not in contact with any of the inner wall surfaces 2a). Thereby, it is possible to prevent the liquid reagent that has reached the first end 1a from flowing out to the second flow path 2 side.

  FIG. 8 shows a conventional structure of the reagent holding unit and the vicinity thereof. In the conventional microchip, the flow path 22b ′ extends from the reagent holding portion 306a and reaches the third substrate 103, and the flow formed by the notch groove provided at the end of the flow path 22b ′ on the third substrate 103 side. Since the first flow path constituted by the path 22c ′ (FIG. 8C) is in contact with the inner wall surface 2a of the second flow path 2, the liquid passing through the flow path 22b ′ is caused by surface tension. , It flows out to the second flow path 2 through the flow path 22c ′.

  Referring to FIG. 7, in the microchip 100 of the present embodiment, when the inner diameter of the first end 1a is φ and the distance from the first end 1a to the inner wall surface 2a facing the first end 1a is r, It is preferable to satisfy the relational expression r> φ / 2, and it is more preferable to satisfy the relational expression r> 3φ / 2. As a result, the liquid reagent swollen outward from the first end 1a due to the surface tension does not contact the inner wall surface 2a facing the first end 1a. The liquid reagent can be more reliably prevented from flowing out.

  The microchip 100 (the outer shape and the fluid circuit structure are as shown in FIGS. 4 to 6) having the structure as shown in FIG. The following liquid reagent holding power test was performed on a microchip similar to the microchip 100 except that the structure in the vicinity of is a structure as shown in FIG. The results are shown in FIG. 18 (n number per graph is 66).

  Liquid reagents were put in each of the reagent holding parts (total of 11) of the microchip 100, the reagent inlet was sealed, and then held at a temperature of 4 ° C. for 240 hours. About the microchip after holding | maintenance, the presence or absence of the outflow of the liquid reagent from the discharge port (1st edge part) in each reagent holding part was confirmed. The same test was repeated a total of 6 times (n = 66), and the outflow rate (100 × number of outflowing reagent holders / 66) was calculated. The liquid reagent retention was performed on three types of liquid reagents having different wettability (contact angles). A similar test was performed on a microchip having the structure shown in FIG.

  As shown in FIG. 18, in the microchip 100 according to the present invention, the outflow rate was 0% even when a liquid reagent with high wettability (low contact angle) was used. On the other hand, in the conventional microchip having the structure shown in FIG. 8, when the liquid reagent having the highest wettability (contact angle of about 41 °) was used, the outflow rate reached about 40%.

  Next, an example of fluid processing using the microchip 100 of the present embodiment will be described with reference to FIGS. 9 to 17 show the state of the liquid (specimen, specific component, liquid reagent, and mixed liquid of the specific component and the liquid reagent) on the upper surface (first substrate side surface) of the second substrate 102 in each step of fluid processing. It is a figure which shows the state of the liquid of a lower surface (3rd board | substrate side surface). (A) in each figure is a figure which shows the state of the liquid of the 2nd board | substrate upper surface (1st fluid circuit), (b) is the state of the liquid of the 2nd board | substrate lower surface (2nd fluid circuit). FIG. 9 to 17 (b), as in FIG. 6, in a state of being reversed left and right so that the correspondence with the upper fluid circuit shown in FIGS. 9 to 17 (a) can be clearly understood. The lower fluid circuit of the second substrate 102 is shown. Further, in the following description, only fluid processing in the fluid circuit of section 1 will be described, but the same processing is performed in other sections, and this can be clearly understood by referring to the drawings. Can do. Furthermore, in the following, a case where the sample is whole blood will be described as an example, but the type of the sample is not limited to this.

(1) Whole blood measurement and liquid reagent measurement step First, in this step, the microchip in the state shown in FIGS. 5 and 6 is directed downward in FIG. 9 (hereinafter simply referred to as downward. FIGS. The same applies to the other directions, and a centrifugal force is applied. Thereby, the whole blood 600 introduced from the sample introduction port 120 (see FIG. 4) of the first substrate 101 is introduced into the sample measuring unit 500 and measured. The whole blood 600 overflowed from the sample measuring section 500 is stored in the overflow liquid storage section 330a [see FIG. 9 (a)]. In addition, by applying the downward centrifugal force, the liquid reagent in the liquid reagent holding portions 301a and 301b passes through the flow paths (through holes) 21b and 21c to the reagent measuring portions 411a and 411b, respectively, and is measured [FIG. 9 (b)]. The liquid reagent overflowing from each liquid reagent metering section is stored in overflow reagent storage sections 331a and 331b in the upper surface side fluid circuit through the flow paths (through holes) 21a and 21d, respectively (see FIG. 9A). ]. At this stage, when there is no liquid volume abnormality with respect to the liquid reagent, the liquid reagent is present in all the overflow reagent storage portions except the overflow reagent storage portion 332b.

(2) Whole blood moving step Next, a rightward centrifugal force is applied. Thereby, the measured whole blood 600 in the sample measuring unit 500 moves to the standby unit 701 of the lower fluid circuit through the through hole 30 (see FIG. 10B).

(3) Blood cell separation step Next, a downward centrifugal force is applied. As a result, the whole amount of the whole blood 600 weighed in the standby unit 701 is introduced into the separation unit 420 through the flow restriction unit 700 (see FIG. 11B). The whole blood 600 introduced into the separation unit 420 is centrifuged at the separation unit 420 and separated into a plasma component (upper layer) and a blood cell component (lower layer). Each liquid reagent is again stored in the reagent metering unit.

(4) Plasma component measurement step Next, a rightward centrifugal force is applied. Thereby, the plasma component separated in the separation unit 420 is introduced into the component measurement unit 401 (and simultaneously introduced into the component measurement units 402, 403, 404, and 405, 406), and is measured [FIG. )reference〕. The plasma component overflowing from the measuring section moves into the upper fluid circuit through the flow path (through hole) 26a [see FIG. 12 (a)].

(5) First mixing step Next, a downward centrifugal force is applied. Thereby, the measured liquid reagent (the liquid reagent held in the reagent holding unit 301a) and the plasma component measured in the component measuring unit 401 are mixed in the reagent measuring unit 411a [first mixing step. First step, see FIG. 13B]. At this time, the liquid reagent remains in the mixing portion 441a of the lower fluid circuit.

  Next, by applying a centrifugal force in the right direction, the mixed solution is further mixed with the liquid reagent remaining in the mixing unit 441a [see the second step of the first mixing step, see FIG. 14B]. These first step and second step are performed a plurality of times as necessary to ensure mixing. Finally, a state similar to the state shown in FIG. 14 is obtained.

(6) Second mixing step Next, an upward centrifugal force is applied. As a result, the mixed liquid in the mixing unit 441a passes through the flow path (through hole) 21e to the mixing unit 441b of the upper fluid circuit, and is held in the other measured liquid reagent (reagent holding unit 301b). The liquid reagent) also reaches the mixing section 441b through the flow path 21e and is mixed (see the second step of the second mixing step, see FIG. 15A).

  Next, by applying a leftward centrifugal force, the mixed solution moves and mixing is promoted as shown in FIG. 16A [second step of the second mixing step, see FIG. 16A. ]. Further, the liquid reagent is accommodated in the overflow reagent accommodating portion 332b by the leftward centrifugal force [see FIG. 16 (a)]. These first step and second step are performed a plurality of times as necessary to ensure mixing. Finally, a state similar to the state shown in FIG. 16 is obtained.

(7) Detection part introduction process Finally, downward centrifugal force is applied. Thereby, the liquid mixture is introduced into the detection unit 311 [refer to FIGS. 17A and 17B for other liquid mixtures as well]. Further, the overflow reagent storage units 331a and 331b and the overflow liquid storage unit 330b are in a state in which liquid reagents or plasma components are stored. The same applies to other overflow reagent storage units. The mixed liquid filled in the detection unit is subjected to optical measurement, and inspection / analysis is performed. For example, a specific component in the mixed liquid can be detected by irradiating light from a direction substantially perpendicular to the surface of the microchip and measuring the transmitted light. At this time, the presence or absence of a plasma component or a liquid reagent is confirmed by irradiating light to the overflow liquid storage portion 330b and each overflow reagent storage portion and measuring the intensity of the reflected light. Confirmation of the presence or absence of the plasma component or liquid reagent is not necessarily performed at this stage, but it is at this stage that the plasma component or liquid reagent can be accommodated in all the overflow reagent storage part and the overflow reagent storage part. Therefore, in order to simplify the operation, it is preferable to confirm the presence or absence of plasma components or liquid reagents after the detection unit introduction step.

<Second Embodiment>
FIG. 19 is a top view showing another example of the microchip of the present invention. A microchip 200 shown in FIG. 19 has a single-layer fluid circuit formed by laminating and bonding a second substrate (not shown in FIG. 19) on a first substrate 1000 having a groove on the surface. It is a microchip. The first substrate 1000 is bonded onto a second substrate (not shown) so that the groove forming side surface faces the second substrate. FIG. 19 shows the surface of the first substrate 1000 opposite to the groove-forming surface, but the groove pattern is shown by a solid line for convenience of explanation. In the microchip 200, the second substrate has the same or similar contour shape as the first substrate 1000. The first substrate 1000 and the second substrate are respectively a transparent substrate and a black substrate made of a thermoplastic resin, for example.

  The microchip 200 is a sample tube mounting unit 1001 for incorporating a sample tube such as a capillary containing whole blood collected from a subject; for separating the whole blood derived from the sample tube into a blood cell component and a plasma component Separation unit 1002; blood cell measurement unit 1003 for measuring separated blood cell components; three reagent holding units 1004, 1005 and 1006 for holding liquid reagents; provided adjacent to reagent holding units 1005 and 1006, respectively Reagent storage units 1007 and 1008 for temporarily storing a liquid reagent; three reagent measuring units 1009, 1010 and 1011 for measuring a liquid reagent; a first unit for mixing blood cell components and a liquid reagent 1 mixing unit 1012; a mixed liquid measuring unit 1013 for measuring a mixed solution of a blood cell component and a liquid reagent; A second mixing unit 1014 for mixing the liquid mixture of the liquid reagent and the other liquid reagent; and a detection unit 1015 for performing inspection / analysis on the finally obtained liquid mixture. Configured.

  The three reagent holding units 1004, 1005, and 1006 have reagent introduction ports 1016, 1017, and 1018 for injecting a liquid reagent into the reagent holding unit, respectively. The reagent introduction ports 1016, 1017, and 1018 are through-holes that penetrate the first substrate 1000 in the thickness direction. The microchip 200 of the present embodiment is normally used after injecting a liquid reagent from the reagent inlets 1016, 1017, and 1018 and then sealing these reagent inlets with a sealing label or the like. . Hereinafter, the liquid reagents that are injected and held in the reagent holding units 1004, 1005, and 1006 through the reagent introduction port will be referred to as liquid reagents R0, R1, and R2, respectively.

  As described above, the fluid circuit included in the microchip 200 of the present embodiment mixes the liquid reagents R0, R1, and R2 in this order with the blood cell components separated from the whole blood, and the obtained mixed liquid. The configuration is suitable for performing inspection and analysis such as optical measurement.

  Here, in the microchip 200 of the present embodiment, each reagent holding part and the structure in the vicinity thereof have the above-described characteristics according to the present invention. The reagent holding unit 1006 will be described as an example. FIG. 20 is a sectional view schematically showing the structure of the reagent holding unit 1006 and the vicinity thereof. In this cross-sectional view, a second substrate 1100 stacked on the first substrate 1000 and a sealing label 1200 for sealing an opening such as a reagent introduction port are also shown.

  As shown in FIG. 20, the reagent holding unit 1006 has one end (second end) connected to the reagent holding unit 1006 to guide the liquid reagent in the reagent holding unit 1006 to the reagent storage unit 1008. The flow path 1006a that penetrates the first substrate 1000 in the thickness direction is provided (similarly, the reagent holding portions 1004 and 1005 also have flow paths 1004a and 1005a that penetrate the first substrate 1000 in the thickness direction, respectively. (See FIG. 19). The channel 1006a corresponds to the first channel described above. The flow path 1006a is such that the other end of the first end 1a (liquid reagent discharge port) is separated from any of the inner wall surfaces 2a of the second flow path 2 (including the reagent storage unit 1008). (In other words, the first end 1a is not in contact with any inner wall surface 2a). Thereby, it is possible to prevent the liquid reagent that has reached the first end 1a from flowing out to the second flow path 2 side.

  An example of fluid processing using the microchip 200 shown in FIG. 19 is roughly as follows. First, a sample tube from which a whole blood sample is collected is inserted into the sample tube mounting unit 1001. Next, a centrifugal force is applied to the microchip in the leftward direction in FIG. 19 (hereinafter, simply referred to as “leftward”, the same applies to the other directions below), and the whole blood sample in the sample tube is taken out, and then downward The whole blood sample is introduced into the separation unit 1002 by centrifugal force and centrifuged to separate the plasma component and the blood cell component. Next, leftward centrifugal force is applied to remove the plasma component in the upper layer. At this time, the removed plasma component is accommodated in the region a. Next, by applying a downward centrifugal force, the blood cell component liquid level in the separation unit 1002 is adjusted, and the removed plasma component is moved to the region b. Next, a rightward centrifugal force is applied, the liquid reagent R0 in the reagent holding unit 1004 is introduced into the reagent measuring unit 1009, and measurement is performed. By this centrifugal force, the liquid reagent R1 in the reagent holding unit 1005 and the liquid reagent R2 in the reagent holding unit 1006 move to the reagent storage units 1007 and 1008, respectively. Further, due to the centrifugal force, the blood cell component in the separation unit 1002 is introduced into the blood cell measuring unit 1003 and measured.

  Next, a downward centrifugal force is applied, and the weighed blood cell component and the liquid reagent R0 are mixed in the first mixing unit 1012 to obtain a mixed solution. With this centrifugal force, the liquid reagent R2 in the reagent storage unit 1008 is measured by the reagent measuring unit 1011. Next, right, downward, leftward, and downward centrifugal forces are sequentially applied to sufficiently mix the liquid mixture. Note that the liquid reagent R1 in the reagent storage unit 1007 is measured by the reagent measuring unit 1010 by applying the leftward centrifugal force. Further, the weighed liquid reagent R1 moves to the second mixing unit 1014 by the last downward centrifugal force.

  Next, after applying a leftward centrifugal force, a leftward upward centrifugal force and then a leftward centrifugal force are applied, and the supernatant of the mixed liquid in the first mixing unit 1012 is introduced into the mixed liquid measuring unit 1013 for measurement. . Next, by applying a downward centrifugal force, the measured mixed liquid and the liquid reagent R1 are mixed in the second mixing unit 1014. Next, leftward and downward centrifugal forces are sequentially applied to sufficiently mix the liquid mixture. In a state where the downward centrifugal force is applied, the measured liquid reagent R2 is located in the region c. Next, a rightward centrifugal force is applied to mix the mixed solution and the liquid reagent R2 in the detection unit 1015, and a downward centrifugal force is further applied to perform sufficient mixing. Finally, a rightward centrifugal force is applied, the mixed solution is accommodated in the detection unit 1015, light is irradiated to the detection unit 1015, and the intensity of the transmitted light is measured.

<Third Embodiment>
21 to 22 are cross-sectional views schematically showing other examples of the microchip of the present invention, and an enlarged portion where the first flow path 1 and the second flow path 2 according to the present invention are provided. It is a figure shown. The microchips of FIGS. 21 to 22 have a laminated structure of the first substrate 7, the second substrate 6, and the third substrate 5 as in the first embodiment, and have a two-layer fluid circuit. Yes. As shown in FIG. 22, as long as the first end portion 1a is separated (not in contact) with any of the inner wall surface 2a of the second flow path 2, the grooves constituting the fluid circuit are the second It can be formed not only on the first substrate 6 but also on the first substrate 7 and the third substrate 5 sandwiching the substrate 6. Also in the microchip having a single-layer fluid circuit as in the second embodiment, a groove can be formed in the other substrate.

<Fourth Embodiment>
The present invention is not limited to the case where the reagent holding portion and the structure in the vicinity thereof have the above-described features according to the present invention. For example, the above-described characteristics according to the present invention may be added to various measuring units such as a component measuring unit that measures a plasma component separated from whole blood shown in FIG. 23 and the vicinity thereof. 23 to 25 are a top view and a cross-sectional view schematically showing another example of the microchip of the present invention, in which a component measuring unit for measuring a plasma component and its vicinity are enlarged. (A) of FIGS. 23-25 is a top view, (b) is sectional drawing. The top view here is a top view of the second substrate 6 on which grooves constituting the fluid circuit are formed.

  The microchip shown in FIG. 23 is a microchip having a laminated structure of the first substrate 7, the second substrate 6, and the third substrate 5, and having two layers of fluid circuits. It has a component measuring unit 2001 for measuring the plasma component 2000 separated in the separating unit (not shown). An opening 2002 is formed on the bottom surface of the component measuring unit 2001, and the first flow path 1 penetrating the second substrate 6 in the thickness direction is connected to the opening 2002. The first flow path 1 is a flow path for guiding the plasma component overflowed during measurement to a waste liquid tank (not shown) in the lower fluid circuit. The first flow path 1 has a first end 1a which is the other end (a discharge port for the overflowed plasma component to the second flow path), which is the second flow path of the lower fluid circuit. The two inner wall surfaces are arranged so as to be separated from each other (that is, the first end portion 1a is not in contact with any inner wall surface). Thereby, since the measured plasma component can be prevented from flowing out to the second flow path 2 side due to the surface tension, the measurement accuracy can be improved.

  An example of the measurement operation of the plasma component using the microchip shown in FIG. 23 will be described with reference to FIGS. First, by applying a centrifugal force in the direction of the arrow shown in FIG. 23, the plasma component 2000 separated in the separation unit (not shown) is introduced into the component measuring unit 2001 (plasma introduction step, FIG. 23), Plasma component measurement is performed by filling component measurement unit 2001 with plasma component 2000 (plasma measurement step, FIG. 24). In the plasma measurement process, the excess plasma component 2000 that exceeds the capacity of the component measurement unit 2001 passes through the first flow path 1 and then the second flow path 2, and the waste fluid tank (not shown) of the lower fluid circuit. ). Since the first flow path 1 and the second flow path 2 have the structure according to the present invention, when the application of centrifugal force is stopped after the plasma measurement step, the measured plasma component is It does not flow out to the second flow path 2 side due to the tension. Finally, the measured plasma component is discharged from the component measuring unit 2001 by applying a centrifugal force in the direction of the arrow shown in FIG. 25 (plasma discharge step, FIG. 25). The discharged plasma component is used for mixing with a liquid reagent.

  DESCRIPTION OF SYMBOLS 1 1st flow path, 1a 1st edge part in 1st flow path, 2nd flow path, 2a Inner wall surface of 2nd flow path, 4 Reagent holding part, 5 3rd board | substrate, 6 1st 2 substrate, 7 first substrate, 11a, 11b, 16a, 16b, 21a, 21b, 21c, 21d, 21e, 22b, 22b ′, 26a, 30 flow path, 100 microchip, 101 first substrate, 102 Second substrate, 103 Third substrate, 110 Reagent introduction port, 120 Sample introduction port, 200 microchip, 301a, 301b, 302a, 302b, 303a, 303b, 304a, 304b, 305a, 305b, 306a Reagent holding unit, 311, 312, 313, 314, 315, 316 detection unit, 330 a, 330 b overflow liquid storage unit, 331 a, 331 b, 332 a, 332 b, 333 a, 33b, 334a, 334b, 335a, 335b, 336a Overflow reagent storage unit, 401, 402, 403, 404, 405, 406 Component metering unit, 411a, 411b, 412a, 412b, 413a, 413b, 414a, 414b, 415a, 415b , 416a Reagent measuring unit, 420 Separating unit, 441a, 441b Mixing unit, 500 Sample measuring unit, 600 Whole blood, 700 Flow rate limiting unit, 701 Waiting unit, 1000 First substrate, 1001 Sample tube mounting unit, 1002 Separating unit , 1003 Blood cell measuring unit, 1004, 1005, 1006 Reagent holding unit, 1007, 1008 Reagent storage unit, 1009, 1010, 1011 Reagent measuring unit, 1012 first mixing unit, 1013 mixed liquid measuring unit, 1014 second mixing unit 1015 detector, 1016,1 17,1018 reagent inlet, 1100 a second substrate, the label 1200 sealing, 2000 plasma component, 2001 component metering unit, 2002 opening.

Claims (3)

A microchip comprising a fluid circuit comprising a space formed therein, and moving a liquid present in the fluid circuit to a desired position in the fluid circuit by centrifugal force ;
The fluid circuit includes a reagent holding unit that stores a liquid reagent, and a first flow having a smaller cross-sectional area than the reagent holding unit that is connected to the reagent holding unit and passes the liquid reagent stored in the reagent holding unit. A second channel having a cross-sectional area larger than that of the first channel that is connected to the first channel and passes the liquid that has passed through the first channel ;
The reagent holding part is surrounded by an upper surface, a bottom surface, and a side surface connecting the upper surface and the bottom surface,
The first channel is connected to the reagent holding unit through an opening defined by a part of the upper surface and a part of the side surface, and is an end on the second channel side. A first end located in the second flow path and spaced from the inner wall surface of the second flow path ;
The reagent holding portion, a microchip the side directed from the bottom to the opening that has an inclined shape. The first channel extends from the upper surface side of the reagent holding unit toward the bottom surface side,
The microchip according to claim 1, wherein the second channel is formed at a position lower than the bottom surface. The relational expression r> φ / 2 is satisfied when the inner diameter of the first end is φ and the distance from the first end to the inner wall of the second flow path is r. Or the microchip of 2.

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